Identification of myosin II as a cripto binding protein and regulator of cripto function in stem cells and tissue regeneration.

Cripto regulates stem cell function in normal and disease contexts via TGFbeta/activin/nodal, PI3K/Akt, MAPK and Wnt signaling. Still, the molecular mechanisms that govern these pleiotropic functions of Cripto remain poorly understood. We performed an unbiased screen for novel Cripto binding proteins using proteomics-based methods, and identified novel proteins including members of myosin II complexes, the actin cytoskeleton, the cellular stress response, and extracellular exosomes. We report that myosin II, and upstream ROCK1/2 activities are required for localization of Cripto to cytoplasm/membrane domains and its subsequent release into the conditioned media fraction of cultured cells. Functionally, we demonstrate that soluble Cripto (one-eyed pinhead in zebrafish) promotes proliferation in mesenchymal stem cells (MSCs) and stem cell-mediated wound healing in the zebrafish caudal fin model of regeneration. Notably, we demonstrate that both Cripto and myosin II inhibitors attenuated regeneration to a similar degree and in a non-additive manner. Taken together, our data present a novel role for myosin II function in regulating subcellular Cripto localization and function in stem cells and an important regulatory mechanism of tissue regeneration. Importantly, these insights may further the development of context-dependent Cripto agonists and antagonists for therapeutic benefit.

Transplanted Human Neural Progenitor Cells Attenuate Motor Dysfunction and Lengthen Longevity in a Rat Model of Ataxia.

The spastic Han Wistar (sHW) rat serves as a model for human ataxia presenting symptoms of motor deterioration, weight loss, shortened lifespan, and Purkinje neuron loss. Past studies revealed that human neural progenitor cells (NPCs) improved ataxic symptoms at 20 d posttransplantation in sHW rats. In this study, we investigated the fate and longer-term effectiveness of these transplanted NPCs. Rats were placed into four treatment groups: an untreated normal control group (n = 10), an untreated mutant rat control (n = 10), a mutant group that received an injection of dead NPCs (n = 9), and a mutant group that received live NPCs (n = 10). Bilateral cerebellar injections containing 500,000 of either live or dead NPCs were performed on mutant sHW rats at 40 d of age. Motor activity for all mutant rats started to decline in open field testing around day 35. However, at day 45, the live NPC-treated mutants exhibited significant improvements in open field activity. Similar improvements were observed during rotarod testing and weight gain through the completion of the experiments (100 d). Immunohistochemistry revealed few surviving human NPCs in the cerebella of 80- and 100-d-old NPC-treated mutants; while cresyl violet staining revealed that live NPC-treated mutants had significantly more surviving Purkinje neurons compared to mutants that were untreated or received dead NPCs. Direct stereotactic implantation of NPCs alleviated the symptoms of ataxia, acting as a neuroprotectant, supporting future clinical applications of these NPCs in the areas of ataxia as well as other neurodegenerative diseases.

Secretomes from metastatic breast cancer cells, enriched for a prognostically unfavorable LCN2 axis, induce anti-inflammatory MSC actions and a tumor-supportive premetastatic lung.

Cancer metastasis is responsible for the clear majority of cancer-related deaths. Survival and expansion of cancer cells at secondary sites requires that these premetastatic microenvironments be primed by primary tumor cells and their secreted factors. Efforts to date have been limited by immune-deficient in vivo models and/or the need for finely-tuned analysis time points that reduce contributions from early-disseminating cancer cells. In this regard, we developed a tumor cell-free syngeneic breast cancer model for characterizing tumor cell secretome-mediated reprogramming of premetastatic tissues. We demonstrate that secretomes from metastatic breast cancer cells differentially regulate the lung and brain, promoting a tumor-supportive lung microenvironment with both elevated CD73 expression and decreased TNFα expression. Using in vitro models of CD73-positive mesenchymal stem cells (MSCs) and macrophages/monocytes, we tested whether MSCs can mediate anti-inflammatory effects of metastatic breast cancer cells. Notably, conditioned media from metastatic Py230 cells reprogrammed the secretomes of MSCs toward an anti-inflammatory state. Mining transcriptome data from Py8119 and Py230 cells revealed a lipocalin 2 (LCN2) axis that is selectively expressed in the metastatic Py230 cells, predicts poor breast cancer patient survival and is elevated in circulating serum of mice chronically treated with conditioned media from Py230 cells. Taken together, these results establish the utility of an immune-competent tumor cell-free model for characterizing the mechanisms of breast cancer cell priming of the premetastatic niche, demonstrate that MSCs can mediate the anti-inflammatory effects of metastatic breast cancer cells and substantiate LCN2 as a promising therapeutic target for blocking breast cancer progression.

Effective Treatment of Traumatic Brain Injury in Rowett Nude Rats with Stromal Vascular Fraction Transplantation.

Traumatic brain injury (TBI) affects 1.9 million Americans, including blast TBI that is the signature injury of the Iraq and Afghanistan wars. Our project investigated whether stromal vascular fraction (SVF) can assist in post-TBI recovery. We utilized strong acoustic waves (5.0 bar) to induce TBI in the cortex of adult Rowett Nude (RNU) rats. One hour post-TBI, harvested human SVF (500,000 cells suspended in 0.5 mL lactated Ringers) was incubated with Q-Tracker cell label and administered into tail veins of RNU rats. For comparison, we utilized rats that received SVF 72 h post-TBI, and a control group that received lactated Ringers solution. Rotarod and water maze assays were used to monitor motor coordination and spatial memories. Rats treated immediately after TBI showed no signs of motor skills and memory regression. SVF treatment 72 h post-TBI enabled the rats maintain their motor skills, while controls treated with lactated Ringers were 25% worse statistically in both assays. Histological analysis showed the presence of Q-dot labeled human cells near the infarct in both SVF treatment groups; however, labeled cells were twice as numerous in the one hour group. Our study suggests that immediate treatment with SVF would serve as potential therapeutic agents in TBI.

Validation of Acoustic Wave Induced Traumatic Brain Injury in Rats.

BACKGROUND: This study looked to validate the acoustic wave technology of the Storz-D-Actor that inflicted a consistent closed-head, traumatic brain injury (TBI) in rats. We studied a range of single pulse pressures administered to the rats and observed the resulting decline in motor skills and memory. Histology was observed to measure and confirm the injury insult. METHODS: Four different acoustic wave pressures were studied using a single pulse: 0, 3.4, 4.2 and 5.0 bar (n = 10 rats per treatment group). The pulse was administered to the left frontal cortex. Rotarod tests were used to monitor the rats' motor skills while the water maze test was used to monitor memory deficits. The rats were then sacrificed ten days post-treatment for histological analysis of TBI infarct size. RESULTS: The behavioral tests showed that acoustic wave technology administered an effective insult causing significant decreases in motor abilities and memory. Histology showed dose-dependent damage to the cortex infarct areas only. CONCLUSIONS: This study illustrates that the Storz D-Actor effectively induces a repeatable TBI infarct, avoiding the invasive procedure of a craniotomy often used in TBI research.

Transplantation of Human Neural Progenitor Cells Reveals Structural and Functional Improvements in the Spastic Han-Wistar Rat Model of Ataxia.

The use of regenerative medicine to treat nervous system disorders like ataxia has been proposed to either replace or support degenerating neurons. In this study, we assessed the ability of human neural progenitor cells (hNPCs) to repair and restore the function of dying neurons within the spastic Han-Wistar rat (sHW), a model of ataxia. The sHW rat suffers from neurodegeneration of specific neurons, including cerebellar Purkinje cells and hippocampal CA3 pyramidal cells leading to the observed symptoms of forelimb tremor, hind-leg rigidity, gait abnormality, motor incoordination, and a shortened life span. To alleviate the symptoms of neurodegeneration and to replace or augment dying neurons, neuronal human progenitor cells were implanted into the sHW rats. At 30 d of age, male sHW mutant rats underwent subcutaneous implantation of an Alzet osmotic pump that infused cyclosporine (15 mg/kg/d) used to suppress the rat's immune system. At 40 d, sHW rats received bilateral injections (500,000 cells in 5 µL media) of live hNPCs, dead hNPCs, live human embryonic kidney cells, or growth media either into the cerebellar cortex or into the hippocampus. To monitor results, motor activity scores (open-field testing) and weights of the animals were recorded weekly. The sHW rats that received hNPC transplantation into the cerebellum, at 60 d of age, displayed significantly higher motor activity scores and sustained greater weights and longevities than control-treated sHW rats or any hippocampal treatment group. In addition, cerebellar histology revealed that the transplanted hNPCs displayed signs of migration and signs of neuronal development in the degenerated Purkinje cell layer. This study revealed that implanted human progenitor cells reduced the ataxic symptoms in the sHW rat, identifying a future clinical use of these progenitor cells against ataxia and associated neurodegenerative diseases.

Efficacy of Two Delivery Routes for Transplanting Human Neural Progenitor Cells (NPCs) Into the Spastic Han-Wistar Rat, a Model of Ataxia.

An emerging avenue for recalcitrant neurodegenerative disease treatment is neural progenitor cell (NPC) transplantation. In this study, we investigated the effectiveness of two different delivery routes of human-derived NPC inoculation: injection into the common carotid artery or unilateral stereotactic implantation into the degenerating cerebellum and hippocampus of spastic Han-Wistar (sHW) rats, a model of ataxia. At 30 days of age, sHW mutants were implanted with osmotic pumps preloaded with cyclosporine. Ten days after pump implantation, the animals were given either 3,000,000 live human-derived NPCs (hNPCs; n = 12) or 3,000,000 dead NPCs (dNPCs; n = 12) injected into the common carotid artery, or were given two unilateral implantations of 500,000 hNPCs into the cerebellum and 500,000 hNPCs into the hippocampus of each sHW rat (n = 12) or 500,000 dNPCs by unilateral implantation into the cerebellum and hippocampus (n = 12). We also compared treated sHW rats to untreated sHW rats: normal rats (n = 12) and sibling sHW rats (n = 12). Motor activity and animal weights were monitored every 5 days to ascertain effectiveness of the two types of delivery methods compared to the untreated mutant and normal animals. Mutant rats with hNPC implantations, but not dNPC or carotid artery injections, showed significant deceleration of motor deterioration (p < 0.05). These mutants with hNPC implantations also retained weight longer than dNPC mutants did (p < 0.05). At the end of the experiment, animals were sacrificed for histological evaluation. Using fluorescent markers (Qtracker) incorporated into the hNPC prior to implantation and human nuclear immunostaining, we observed few hNPCs in the brains of carotid artery-injected mutants. However, significant numbers of surviving hNPCs were seen using these techniques in mutant cerebellums and hippocampi implanted with hNPC. Our results show that direct implantation of hNPCs reduced ataxic symptoms in the sHW rat, demonstrating that stereotactic route of stem cell delivery correlates to improved clinical outcomes.

Muscle-specific changes in length-force characteristics of the calf muscles in the spastic Han-Wistar rat.

The purpose of the present study was to investigate muscle mechanical properties and mechanical interaction between muscles in the lower hindlimb of the spastic mutant rat. Length-force characteristics of gastrocnemius (GA), soleus (SO), and plantaris (PL) were assessed in anesthetized spastic and normally developed Han-Wistar rats. In addition, the extent of epimuscular myofascial force transmission between synergistic GA, SO, and PL, as well as between the calf muscles and antagonistic tibialis anterior (TA), was investigated. Active length-force curves of spastic GA and PL were narrower with a reduced maximal active force. In contrast, active length-force characteristics of spastic SO were similar to those of controls. In reference position (90° ankle and knee angle), higher resistance to ankle dorsiflexion and increased passive stiffness was found for the spastic calf muscle group. At optimum length, passive stiffness and passive force of spastic GA were decreased, whereas those of spastic SO were increased. No mechanical interaction between the calf muscles and TA was found. As GA was lengthened, force from SO and PL declined despite a constant muscle-tendon unit length of SO and PL. However, the extent of this interaction was not different in spastic rats. In conclusion, the effects of spasticity on length-force characteristics were muscle specific. The changes observed for GA and PL muscles are consistent with the changes in limb mechanics reported for human patients. Our results indicate that altered mechanics in spastic rats cannot be attributed to differences in mechanical interaction, but originate from individual muscular structures.

Neuroprotective effects of moderate aerobic exercise on the spastic Han-Wistar rat, a model of ataxia.

Research has shown that physical exercise may reduce degeneration in certain brain regions experiencing ataxia. Our laboratory utilized mutant spastic Han-Wistar rats (sHW) that display developmental abnormalities, including spastic paresis, fore limb tremors, hind limb rigidity, and a reduced life span (60-65 days of age). Concomitant neurodegeneration has been observed in the cerebellum (Purkinje cells). The purpose of this study was to investigate if moderate, aerobic exercise could reduce Purkinje cell neurodegeneration and improve the motor ability and survival of the mutant sHW rat. Mutant male littermates at the ages of 20 (n=11 pairs) and 30 (n=13 pairs) days old were divided into running groups and non-running groups. Mutant rats were run on a motorized treadmill at the rate of 15 m/min with a 10% slope. The "running" group ran for 30 min per day, 5 days a week; the "non-runners" remained nearby in the training facility. These conditions were held constant until the mutant runners could no longer run due to disease progression. Moderate exercise increased the lifespan of running mutant rats in both the 20-day start group (14% increase) and 30-day start group (13% increase). The rats exhibited improved motor function as open-field tests showed higher activity scores for runners after 50 days. Histological examination of the cerebellum revealed a 62% increase in Purkinje cell survival of the runners. These results suggest that aerobic exercise ameliorates, at least partially, cerebellar dysfunction in the sHW rat, an excellent model of ataxia.